The content of the publications referred to in the following discussion is in each case incorporated herein by reference in all respects relevant to the understanding and performance of the present invention.
Angiogenesis is a significant underlying pathological process in many, if not all, of the major human and other mammalian diseases of the western world. In addition to cancer, for example, it is a prominent feature of vascular disease (atheromatous plaque development requires proliferation of the vaso vasorum), diabetes, rheumatoid arthritis, proliferative retinopathy. The predominance of angiogenesis within clinical pathologies and the relatively limited role in normal physiology (in comparison to cell division) underpins the proposal that anti-angiogenic therapy may be an effective and generally well tolerated treatment modality, particularly but not exclusively in patients with life threatening disease.
VEGF pre-mRNA is differentially spliced from 8 exons to form mRNAs encoding at least six proteins (38) that have been widely studied and accepted as pro-angiogenic pro-permeability vasodilators. The most studied form has 165 amino acids in final structure and is termed VEGF165. In 2002, an alternative isoform, VEGF165b, was identified (2 and WO-A-03/012105), generated by differential splice acceptor site selection in the 3′UTR of exon 8 of the VEGF gene (see FIG. 1A of the accompanying drawings), thus resulting in two sub-exons, termed exon 8a (previously called exon 8) and exon 8b (previously called exon 9), in keeping with the nomenclature for exon 6 (24). Distal splicing into the splice acceptor site for exon 8b results in an open reading frame of 18 bases. The open reading frames of Exons 8a and 8b both code for 6 amino acids; exon 8a for Cys-Asp-Lys-Pro-Arg-Arg and exon 8b for Ser-Leu-Thr-Arg-Lys-Asp. This alternative splicing predicted an alternate family of VEGF isoforms (complementary to the existing isoforms) expressed as multiple proteins in human cells and tissues, of which VEGF121b, VEGF165b, VEGF145b and VEGF189b have now identified (42). The family has been termed VEGFxxxb where xxx is the amino acid number (see FIG. 1B of the accompanying drawings). This alternate C terminus enables VEGF165b to inhibit VEGF165 induced endothelial proliferation, migration, vasodilatation (2), and in vivo angiogenesis and tumour growth (54). These effects were shown to be specific for VEGF, since VEGF165b does not affect fibroblast growth factor (FGF)-induced endothelial cell growth or proliferation. Moreover, unlike all other VEGF isoforms, VEGF165b is down-regulated in cancers so far investigated, including renal cell (2), prostate (54) and colon carcinoma (52) and malignant melanoma (44). Furthermore, VEGF splice variant expression is altered in angiogenic microvascular phenotypes of proliferative eye disease and pre-eclampsia (3, 42). The receptor binding domains are still present in VEGF165b, and hence it acts as a competitive inhibitor of VEGF165; it binds to the receptor but does not stimulate the full tyrosine phosphorylation of the VEGFR activated by VEGF165 (4, 54). This isoform therefore appears to be an endogenous anti-angiogenic agent formed by differential splicing.
The nomenclature, described above, of the alternative splice variants of the C terminal exon 8 of the VEGF gene, which identifies as exon 8a the portion of exon 8 adjacent the proximal splice site and coding for the aminoacid sequence CDKPRR (SEQ. ID. No. 1) and as exon 8b the portion of exon 8 adjacent the distal splice site and coding for the amino-acid sequence SLTRKD (SEQ. ID. No. 2), will be used herein in place of the older nomenclature used, for example, in WO-A-03/012105 (see FIG. 4a of WO-A-03/012105, which Figure is hereby specifically incorporated herein by reference), which identified the exon coding for SEQ. ID. No. 1 as exon 8 and the exon coding for SEQ. ID. No. 2 as “exon 9”.
Alternative splicing is a mechanism of differential gene expression that allows inclusion or substitution of specific exons and the production of structurally and functionally distinct proteins from a single coding sequence (27, 48). It is widely accepted that alternative splicing of VEGF that results in the conventional pro-angiogenic family is fundamental to the regulation of its bioavailability; the inclusion and exclusion of exons 6 and 7, which contain the heparin binding domains, affect the generation of diffusible proteins (24). Moreover, VEGF gene expression is transcriptionally regulated by a diversity of factors including hypoxia (47), growth factors and cytokines (18) (29) (40) (1), hormones (49, 8), oncogenes and tumour suppressor genes (45, 37). Despite the importance of the pro- and anti-angiogenic VEGF isoforms in regulating the angiogenic ‘switch’ in a wide variety of disease states, and in contrast to the well described regulation of mRNA VEGF transcription, almost nothing is known about the molecular and cellular pathways that regulate alternative splicing of VEGF in general and C′terminal splicing in particular. Of the more than 18,000 manuscripts published on VEGF, only three have investigated regulation of splicing, namely (30, 9, 12).
mRNA splicing occurs during transcription (36) mediated by splicing proteins which form the spliceosome (5). Splicing is regulated, however, by splicing regulatory factors (SRFs). These include the Serine/arginine Residue (SR) proteins (e.g. 9G8, SF2/ASF, Srp40, Srp55, etc) that regulate binding to exon splicing enhancers (ESEs) in the mRNA (5).
General mechanisms of splicing can be regulated genetically: base sequences in the transcript influence splice factor affinities (33), and promoters can also influence alternative splicing through the selective recruitment of splice factors to the C terminal domain of RNA polymerase 1 (23). It is also clear now that signal transduction pathways can influence alternative splicing in response to environmental cues (13, 26). A sequence mutation in tumours could theoretically result in altered splicing. However, as splicing from VEGF165 to VEGF165b has previously been demonstrated upon differentiation of glomerular epithelial cells (11), this splicing is unlikely to be mutation-dependent.
Pre-mRNA has intronic and exonic sequences that bind SRFs—splicing enhancers and silencers (22). Exon splicing depends upon the balance of activities of SR proteins, including SF2/ASF, Srp40, Srp55, and 9G8. Furthermore growth factors such as Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factor-β1 (TGF-β1) and Platelet-derived Growth Factor (PDGF) have previously been shown to increase total VEGF expression, but their effect on terminal exon splice site selection was previously unknown (16, 18, 31, 34).
We have investigated the potential role of SR splicing factors, kinases, and growth factors in VEGF terminal exon splicing.
The present invention is based in one aspect on our surprising finding from this work, that agents which cause distal splice site (DSS) selection in exon 8 of VEGF to be favoured (see FIGS. 1 and 4a of WO-A-03/012105 and FIG. 1 of the accompanying drawings) can play an important role in regulating the alternative splicing in favour of the anti-angiogenic VEGFxxxb isoforms, an effect which appears to be general to all the VEGF isoforms so far studied, from which we have established that VEGF-exon-8-DSS enhancers and/or VEGF-exon-8-PSS (proximal splice site) inhibitors and their in vivo activators, upregulators and potentiators, modified forms of any of the foregoing having a secondary functionality useful for control of their primary activity or the effects thereof, and expression vector systems for expressing any of the foregoing agents in vivo, will have valuable therapeutic activities in anti-angiogenic therapies.
The present invention is based in another aspect on our surprising finding from this work, that agents which cause PSS selection in exon 8 of VEGF to be favoured (see FIGS. 1 and 4a of WO-A-03/012105 and FIG. 1 of the accompanying drawings) can play an important role in regulating the alternative splicing in favour of the pro-angiogenic VEGFxxx isoforms, an effect which appears to be general to all the VEGF isoforms so far studied, from which we have established that VEGF-exon-8-PSS enhancers and/or VEGF-exon-8-DSS inhibitors and their in vivo activators, upregulators and potentiators, modified forms of any of the foregoing having a secondary functionality useful for control of their primary activity or the effects thereof, and expression vector systems for expressing any of the foregoing agents in vivo, will have valuable therapeutic activities in pro-angiogenic therapies.